55.1, a gene of unknown function of phage T4, impacts on Escherichia coli folate metabolism and blocks DNA repair by the NER

Abstract

SummaryPhage T4, the archetype of lytic bacterial viruses, needs only 62 genes to propagate under standard laboratory conditions. Interestingly, the T4 genome contains more than 100 putative genes of unknown function, with few detectable homologues in cellular genomes. To characterize this uncharted territory of genetic information, we have identified several T4 genes that prevent bacterial growth when expressed from plasmids under inducible conditions. Here, we report on the various phenotypes and molecular characterization of 55.1, one of the genes of unknown function. High-level expression from the arabinoseinducible P BAD promoter is toxic to the bacteria and delays the intracellular accumulation of phage without affecting the final burst size. Low-level expression from T4 promoter(s) renders bacteria highly sensitive to UV irradiation and hypersensitive to trimethoprim, an inhibitor of dihydrofolate reductase. The delay in intracellular phage accumulation requires UvsW, a T4 helicase that is also a suppressor of 55.1-induced toxicity and UV sensitivity. Genetic and biochemical experiments demonstrate that gp55.1 binds to FolD, a key enzyme of the folate metabolism and suppressor of 55.1. Finally, we show that gp55.1 prevents the repair of UV-induced DNA photoproducts by the nucleotide excision repair (NER) pathway through interaction with the UvrA and UvrB proteins.

55.2, a Phage T4 ORFan Gene, Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

“…2 ORFan was deleterious for E . coli growth [ 29 ]. Such plasmids confer an arabinose sensitive phenotype (Ara S ) on the carrier host strain.…”

“…Plasmids from such suppressor strains were purified and the suppressor activity of the library plasmids verified. Non-specific suppressors were identified and eliminated as previously described [ 29 ]. After testing the equivalent of ≈ 180 E .…”

“…Intracellular phage growth assays were performed as previously described [ 29 ] with the following modifications. CR63 bacteria were grown in M9 medium supplemented with 50 μg ml -1 tryptophan and 0.4% glucose without (M9) or with 1% casmino acids (M9S) to a concentration of 1 x 10 8 cfu ml -1 and placed on ice.…”

“…We previously identified several T4 ORFans whose ectopic expression significantly inhibits E . coli growth [ 29 ]. As described above, many DNA transactions require the resolution of DNA topological issues.…”

Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7

“…Phages have coevolved with bacteria for over 3 billion years and have thus developed molecules to specifically and optimally inhibit or divert key metabolic functions. Examples of bacterial targets inhibited by phage-derived products include the δ subunit of the DNA polymerase III clamp loader, inhibited by gene product (Gp) 8 of the coliphage N4 (7); the Staphylococcus aureus putative helicase loader, DnaI, inhibited by ORF104 of bacteriophage 77 (5); a key enzyme of folate metabolism, FolD, inhibited by Gp55.1 of the coliphage T4 (8); and the essential cell-division protein, filamenting temperature-sensitive mutant Z (FtsZ), inhibited by Gp0.4 of the coliphage T7 (9). These examples suggest that there are other phage products that may inhibit other bacterial targets.…”